Vitrified composition which preserves biological materials

ABSTRACT

The present invention relates to a method for preserving biological material comprising the steps of: providing a vitrification solution comprised of the biological material and a vitrification agent where the solution has a temperature in the range from 0.1° C. to 17.9° C.; microwaving the vitrification solution for a first period of time; allowing the vitrification solution to rest for a second period of time; repeating steps b and c until the vitrification solution enters into a glassy state.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/769,249, filed Jun. 27, 2007 now U.S. Pat. No. 7,883,664.

FIELD OF THE INVENTION

The present invention relates to a vitrified composition which preservesbiological materials above cryogenic temperatures.

BACKGROUND OF THE INVENTION

The preservation and storage of biological materials (e.g., mammaliancells) at room temperature, while maintaining the viability of thebiological material, is a long unrealized goal. Existing methodsavailable for preservation and storage of biological materials includecryo-preservation and dehydration. The use of protectant substances toenhance the viability of the biological material after recovery fromstorage is prevalent in both cryo-preservation and dehydration.

Cryo-preservation involves cooling the biological material totemperatures which arrest the material's biochemical and chemicalprocesses. Preservation is maintained as long as the temperature ismaintained at sufficiently low values. Cryo-preservation is currentlythe only technique proven to preserve mammalian biological materialwhile maintaining the material's viability. However, cryo-preservationhas its drawbacks and limitations. Cryo-preservation is expensive andlimits the transportation of biological materials to locations where therequired temperature may be maintained.

Dehydration involves removing water (desiccation) from the biologicalmaterial in order to dramatically limit or arrest the material'sbiochemical and chemical processes. The storage temperature ofdehydrated biological material may be above cryogenic temperature or atroom temperature if the degree of drying is sufficient to arrestprocesses at this temperature. The extent of dehydration required may beextreme. Freeze-drying is an example of the dehydration method, whereinbiological material is cooled to a temperature where ice forms and thenthe sample is subsequently dried under vacuum.

The dehydration of biological material suffers from a major limitationin long-term storage at ambient conditions: the degradation of thebiological material by cumulative chemical stresses encountered as thevitrification solution gets concentrated in the extra-cellular space.This results in irreversible cell damage before the cells and thevitrification solution can reach a suitably low moisture content tobecome glassy. The degradation occurs regardless of the drying modeemployed (dry-box, vacuum, etc).

U.S. Pat. No. 6,808,651 discloses a method for creating a thermoplasticshaped-body by concentrating a trehalose solution. The trehalosesolution is concentrated by heating it to a temperature of at least 165°C. in order to reduce the solution's water content. However, there is nomention of using trehalose to aid in the preservation of any type ofbiological material. Additionally, in the present invention, heating abiological composition to a temperature in excess of 50° C., let alone165° C., will almost certainly cause irreversible damage to thebiological material contained within the composition and render itnon-viable.

The use of microwaves to aid in the dehydration of biological materialhas met with little success. Microwave processing using non-ionizingelectromagnetic radiation can actively induce the evaporation of polarmolecules like water from a sample of biological material. The vibrationof polar molecules in a constantly changing electrical field ofmicrowave radiation rapidly increases the temperature of the sample. Therapid increase in temperature has numerous adverse biological effectsand results in a non-viable sample.

The article, Making Monosaccharide and Disaccharide Sugar Glasses byUsing Microwave Oven, published in the Journal of Non-CrystallineSolids, Volume 333, Issue 1, 1 Jan. 2004, Pages 111-114, discloses amethod for making sugar glass without caramelization of the sugarthrough the use of microwaves. Additionally, the article discloses thedesire to use sugar glass to conduct physical aging studies and studyrelaxation dynamics because of the high glass transition temperature ofthe sugars. The article demonstrates the utility of microwave radiationas a means to quickly remove water from materials. While the articledoes disclose some of the protective characteristics of trehalose onproteins and biomembranes, there is no mention of using microwaveradiation on a variety of vitrification agents, including trehalose, forthe preservation and storage of biological materials above cryogenictemperature, while maintaining the viability of the biological material.

A technology that facilitates dry storage of biological material abovecryogenic temperatures would greatly bolster efforts in cellular andtissue engineering, cell transplantation, and biosensor technology.Hence, there exists an unsatisfied need for a composition and method topreserve and store biological materials above cryogenic temperaturewhile maintaining the material's viability.

SUMMARY OF THE INVENTION

The present invention relates to a method for preserving biologicalmaterial comprising the steps of: providing a vitrification solutioncomprised of the biological material and a vitrification agent where thesolution has a temperature in the range from 0.1° C. to 17.9° C.;microwaving the vitrification solution for a first period of time;allowing the vitrification solution to rest for a second period of time;repeating steps b and c until the vitrification solution enters into aglassy state.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Bulk temperature of a 20 microliter (μl) droplet aftercumulative heating in a microwave. The inset legend indicates theduration of the active heating period. In all cases samples were allowedto cool for 30 seconds (s) between heating periods. This cooling periodis not included in the total time, hence the abscissa reflectscumulative heating time, not cumulative process time. The level ofdryness achieved by the end of the process is shown in brackets,expressed in units of gH₂O/gdw.

FIG. 2. Sample water content as a function of cumulative microwaveexposure time. The microwave was activated for a period of 30 secondsout of every minute. The ambient relative humidity during the experimentwas recorded using a sling psychrometer.

FIG. 3. Viability response of J774 cells that were microwave-processedusing alternating 30 second heating and rest periods, shown as afunction of final moisture content. The viability of detached cells wasdetermined using Trypan Exclusion. The viability of attached cells wasdetermined using Calcein-AM and Propidium Iodide staining. Totalviability was calculated according to Equation (1). The inset shows thetotal number of detached cells as a function of moisture content.

FIG. 4: Viability response of trehalose-treated J774 cells that weremicrowave-processed using alternating 30 second heating and restperiods, shown as a function of final moisture content. The viability ofdetached cells was determined using Trypan Exclusion. The viability ofattached cells was determined using Calcein-AM and Propidium Iodidestaining. Total viability was calculated according to Equation (1). Theinset shows the total number of detached cells as a function of moisturecontent.

FIG. 5: Comparison of adjusted resazurin response of 10000 cells after420 s of microwave treatment [gH2O/gdw=4.34; T=420 s of microwavetreatment, C=Control]. Cells had been exposed to 50 milliMolar (mM)trehalose for 18 hours prior to processing. n=5.

FIG. 6: Bright field images of a 20 μl droplet containing J774 cellsdried to a final moisture content of 4.258 gH2O/gdw. Images (A) and (B)were acquired after drying the droplet for 35 minutes in a desiccationchamber. Images (C) and (D) were acquired following drying in amicrowave oven for 330 s of accumulative heating time (660 s totalprocess time). The bars in the images (A) and (C) represent a length of1320 micrometers (μm) and the bars in the images (B) and (D) represent alength of 268 μm.

FIG. 7: Fluorescence images of regions of 20 μl droplet containing J774cells dried to a final moisture content of ˜4.3 gH₂O/gdw. Image (A) and(B) were acquired after drying the droplet for 35 min in a desiccationchamber (4.258 gH₂O/gdw). Images (C) and (D) were acquired followingdrying with microwave technique for a cumulative 420 s (4.309 gH2O/gdw).Images (A) and (C) are from the central region of the dried droplets andimages (B) and (D) are from the edge of the same droplets. The bars inall the images represent a length of 145 μm.

FIG. 8: Temperature of 20 μL trehalose droplet after cumulativemicrowaving at 600 W. The heating cycle consisted of 15 and 30 secondperiods. In all cases the samples were passively cooled for 30 secondsbefore temperature reading. The data points represent only thecumulative active heating time, and do not include the passive coolingtime intervals.

FIG. 9: Water content of sample as a function of cumulative microwavingtime. The samples were micro waved for 30 seconds and passively cooledfor 30 seconds between each cycle. The relative humidity was measuredbefore and after each experiment using a humidity temperature pen. Thesamples at the end of each experiment were further dried to remove waterfor dry mass assessment.

FIG. 10. Grams of H₂O per grams dry weight over time (minutes) for atrehalose solution. Samples were dried simultaneously at 120 W.

FIG. 11. Temperature (° C.) over time (minutes) for a trehalosesolution. Samples were dried simultaneously at 120 W.

FIG. 12. Water content (mean±SD) in germinal vesicle samples exposed tocumulated microwave pulses after membrane poration and trehaloseexposure.

FIG. 13. Oocyte morphology (A-C), germinal vesicle structure (D-F), andgerminal vesicle DNA integrity or migration (G-I) after exposure todifferent treatments before desiccation.

DETAILED DESCRIPTION

The present invention refers to a biological composition which may bestored above cryogenic temperature and remains viable for laterreanimation. Biological composition, as used herein, refers to a mixturewhich may comprise a vitrification solution and various additionalliquid and solid materials. The biological composition may be comprisedof a vitrification solution in a glassy state. The vitrificationsolution may be comprised of a biological material, a vitrificationagent, and various additional materials.

Additionally, a method is described by which preservation and storagemay be achieved above cryogenic temperature such that the function ofthe biological material may be recovered. In one embodiment of thepresent invention, the method involves the controlled dehydration of avitrification solution (and the cells within this solution) usingmicrowave energy, to create a uniformly dried biological composition viaa rapid and controllable process. The method uses intermittent microwaveenergy to cause a small amount of heating in a vitrification solutioncontaining biological material. This heating pulse is enough to causewater loss from the vitrification solution, but not enough to allow thelocal temperature to rise above 50° C. in the case of biological cellsand tissues, or above the temperature of thermal denaturation in thecase of proteins. By intermittently heating the vitrification solutionin well-timed pulses, extensive dehydration can be achieved withinminutes, thereby minimizing the time the biological material (e.g.,cells, proteins, etc.) are exposed to adverse chemical stresses as thevitrification solution concentrates to the point where it will becomeglassy at the desired temperature. Alternatively temperature overshootcan be controlled by intermittently cycling the power on and off inresponse to direct thermal feedback from temperature probes (ex.Thermocouples or Infrared sensors) directed at the sample.

In another embodiment of the present invention, the method involves thecontrolled dehydration of two or more vitrification solutions (and thecells within each solution) using microwave energy within the samemicrowave, to create a uniformly dried biological composition via arapid and controllable process. The method uses intermittent microwaveenergy to cause a small amount of heating in each vitrification solutioncontaining biological material. This heating pulse is enough to causewater loss from each vitrification solution, but not enough to allow thelocal temperature to rise above 50° C. in the case of biological cellsand tissues, or above the temperature of thermal denaturation in thecase of proteins. By intermittently heating each vitrification solutionin well-timed pulses, extensive dehydration can be achieved withinminutes, thereby minimizing the time the biological material (e.g.,cells, proteins, etc.) are exposed to adverse chemical stresses as eachvitrification solution concentrates to the point where it will becomeglassy at the desired temperature. Alternatively temperature overshootcan be controlled by intermittently cycling the power on and off inresponse to direct thermal feedback from temperature probes (ex.Thermocouples or Infrared sensors) directed at the sample.

Some lower animals and numerous plants are capable of surviving completedehydration. This ability to survive in a dry state (anhydrobiosis)depends on several complex intracellular physiochemical and geneticmechanisms. Among these mechanisms is the intracellular accumulation ofsugars (e.g., saccharides, disaccharides, oligosaccharides) which act asa protectant during desiccation, Trehalose is one example of adisaccharide naturally produced in desiccation tolerant animals.

Sugars may offer protection to desiccation tolerant animals in severaldifferent ways. A sugar molecule may effectively replace ahydrogen-bounded water molecule from the surface of a folded proteinwithout changing its conformational geometry and folding due to theunique placement of the hydroxyl groups on a trehalose molecule. A sugarmolecule may also prevent cytoplasmic leakage during rehydration bybinding with the phospholipid heads of the lipid bilayer. Furthermore,many sugars have a high glass transition temperature, allowing them toform an above cryogenic temperature or a room temperature glass at lowwater content. The highly viscous ‘glassy’ state reduces the molecularmobility, which in turn prevents degradative biochemical reactions thatlead to deterioration of cell function and death.

In unprotected mammalian cells, desiccation stress causes severemembrane damage and denaturation of cellular protein which results incell death. However, through the addition and/or imbibition ofvitrification agents (e.g., sugars) the survival of several desiccationintolerant biological materials may be improved during drying,including, but not limited to, human mesenchymal stem cells, murinefibroblast cells, blood platelets, bacteria, viruses, mammalian cellmembranes, liposomes, and enzymes. To impart its protective effectduring desiccation, the vitrification agent may be present on both sidesof the cell membrane. Generally, the vitrification agent will notpermeate the cell membrane without outside aid. To achieve permeation, avariety of techniques have been explored in order to find an efficientmechanism to introduce vitrification agents inside cells. These includetransfection, engineered pores, microinjection, and endocytosis.

The method described above also offers an additional beneficial effectof uniformity within the biological composition. Classic ambient dryingtechniques (e.g., dry box, vacuum, freeze drying) lead to non-uniformsamples with cells clustered in the water-rich region of thenon-homogeneous vitrification solution. The intermittent microwavemethod described herein may also help prevent the ‘skinning effect’(creation of a glassy outer layer that reduces the rate of water lossfrom the central portion of the sample) that accompanies many classicambient drying methods.

Storable or storage, as used herein, refers to a biologicalcomposition's ability to be preserved and remain viable for use at alater time. Above cryogenic temperature, as used herein, refers to atemperature above −150° C. Above freezing temperature, as used herein,refers to a temperature above 0° C. In one embodiment, above freezingtemperature refers to a temperature range between 1° C. and 17° C. Inanother embodiment, above freezing temperature refers to a temperaturein the range between 0.1° C. and 17.9° C. In still another embodiment,above freezing temperature refers to a temperature range between 0.1 and26° C. Room temperature, as used herein, refers to a temperature rangebetween 18 and 26° C. The duration a biological material may remainviable during storage above cryogenic temperature may vary from onematerial to the next. In one embodiment, a biological material mayremain viable while in storage above cryogenic temperature for 2-20days. In another embodiment, a biological material may remain viablewhile in storage above cryogenic temperature for 10 weeks. In yetanother embodiment, a biological material may remain viable while instorage above cryogenic temperature for up to one year. In yet anotherembodiment, a biological material may remain viable while in storageabove cryogenic temperature for up to 10 years.

Vitrification, as used herein, is a process of converting a materialinto a glass-like amorphous material. The glass-like amorphous solid maybe free of any crystalline structure. Solidification of a vitreous solidoccurs at the glass transition temperature T_(g).

Solution, as used herein, refers to either a homogenous or heterogeneousmixture of solids and liquids. A homogenous solution is one which has adefinite, true composition and properties (e.g., any amount of a givensolution has the same composition and properties as any other amount ofthe same solution). A heterogeneous solution is one that has a definitecomposition, however any amount of a given solution may not have thesame composition any other amount of the same solution. In oneembodiment, a homogenous solution may include a biological material anda vitrification solution wherein the biological material is uniformlydistributed throughout the viatrification solution. In anotherembodiment, a heterogeneous solution may include a biological materialand a vitrification solution wherein the biological material isnon-uniformly distributed throughout the vitrification solution.

Vitrification solution, as used herein, refers to a solution which iscomprised of a vitrification agent and a biological material. Thevitrification solution is capable of vitrifying at its glass transitiontemperature (T_(g)). In one embodiment, the vitrification solution maybe comprised of water, a vitrification agent, and an organ to betransplanted. In another embodiment, the vitrification solution may becomprised of water, a disaccharide, and mammalian cells. In yet anotherembodiment, the vitrification solution may be comprised of trehalose andcellular proteins. In yet another embodiment, the temperature of thevitrification solution may not rise above 50° C. in the case ofbiological cells and tissues, or above the temperature of thermaldenaturation in the case of proteins.

Biological material, as used herein, refers to materials which may beremoved or derived from living organisms. Examples of biologicalmaterials include, but are not limited to, proteins, cells, tissues,organs, cell-based constructs, or combinations thereof. In oneembodiment, biological material may refer to mammalian cells. In yetanother embodiment, biological material may refer to a heart, lungs, orkidney to be used in a transplant procedure. In yet another embodiment,biological material may refer to human mesenchymal stem cells, murinefibroblast cells, blood platelets, bacteria, viruses, mammalian cellmembranes, liposomes, enzymes, or combinations thereof. In still anotherembodiment, biological material may refer to reproductive cellsincluding sperm cells, spermatocytes, oocytes, ovum, embryos, germinalvesicles, or combinations thereof. In yet another embodiment, biologicalmaterial may refer to whole blood, red blood cells, white blood cells,platelets, viruses, bacteria, algae, fungi, or combinations thereof.

Vitrification agent, as used herein, is a material that forms a glass,or that suppresses the formation of crystals in other materials, as themixture cools or densifies. The vitrification agent may also provideosmotic protection or otherwise enable cell survival during dehydration.Vitrification agents include, but are not limited to, dimethylsulfoxide,glycerol, sugars, polyalcohols, methylamines, betaines, antifreezeproteins, synthetic anti-nucleating agents, polyvinyl alcohol,cyclohexanetriols, cyclohexanediols, inorganic salts, organic salts,ionic liquids, or combinations thereof. In one embodiment, thevitrification agent may be any water soluble solution that yields asuitable glass for storage of biological materials. In anotherembodiment, the vitrification agent may be a disaccharide. In yetanother embodiment, the vitrification agent may be trehalose. In yetanother embodiment, the vitrification agent may be imbibed within acell, tissue, or organ.

Glassy state, as used herein, refers to a state of matter. An amorphousmaterial enters its glassy state when it passes below its glasstransition temperature (T_(g)). The glassy state combines someproperties of crystals and some of liquids but remains distinctlydifferent from both. In one embodiment, glassy state may refer to thestate the biological composition enters upon dropping below its glasstransition temperature. In another embodiment, the glassy state mayrefer to the state the vitrification solution and/or vitrification agententers upon dropping below its glass transition temperature. In yetanother embodiment, the glassy state may have the mechanical rigidity ofa crystal, but the random disordered arrangement of molecules thatcharacterizes a liquid.

Water-soluble, as used herein, refers to the ability for a givensubstance, the solute, to dissolve in water, the solvent. In oneembodiment, water-solubility may be measured in terms of the maximumamount of solute dissolved in a solvent at equilibrium. The resultingsolution is called a saturated solution. In another embodiment,water-soluble may refer to the vitrification agent's ability to dissolvein water.

Amorphous material, as used herein, is a material in which there is nolong-range order of the positions of the atoms. Conversely, materials inwhich there is long-range atomic order are called crystalline materialsor morphons. Amorphous materials are often prepared by rapidly coolingmolten material, such as glass. The cooling reduces the mobility of thematerial's molecules before they can pack into a more thermodynamicallyfavorable crystalline state. Additives which interfere with the primaryconstituent's ability to crystallize may produce amorphous material. Inone embodiment, the vitrification solution may be an amorphous material.In another embodiment, the vitrification agent may be an amorphousmaterial. In yet another embodiment, the biological composition may bean amorphous material.

Glass transition temperature (T_(g)), as used herein, is the temperatureabove which amorphous materials (e.g., a vitrification solution) behavelike liquids and below which amorphous materials behave in a mannersimilar to those of a crystalline phase and enters the glassy state.This is not a fixed point in temperature, but is instead variabledependant on the timescale of the measurement used. In one embodiment,the vitrification solution may have a glass transition temperature inthe range of −150° C. to 30° C.

Microwaves, as used herein, refers to electromagnetic waves havingoperating frequencies anywhere from 0.3 GHz to 300 GHz. Microwaves arecommonly used in the food, communication, and chemical industries.Microwaving, as used herein, refers to the use of non-ionizingelectromagnetic radiation to actively induce the evaporation of polarmolecules (e.g., water) from a biological composition. The vibration ofpolar molecules in a constantly changing electrical field of microwaveradiation increases the temperature of the system quickly. Increase oftemperature is perhaps the most important factor associated withmicrowave radiation and the majority of the effects on biologicalmaterials are directly related to the heating effect. The maximum outputpower of the microwave may vary in the range of 1 Watt (W) to 600 W. Inone embodiment, the microwave maximum output power may be 600 W. Inanother embodiment, the microwave maximum output power may be 300 W. Inyet another embodiment, the microwave maximum output power may be 100 W.In yet another embodiment, the microwave maximum output power may be 1W.

Biological materials may endure severe damage at multiple levelsresulting from exposure to high levels of microwave radiation. Damagemay include, but is not limited to, cell membrane degradation, cellularprotein denaturation, damage to the sub-cellular components, orcombinations thereof. The increase in temperature limits the time thebiological material may be continuously exposed to microwave radiation.The duration of microwave exposure is directly related to the heatgenerated by the microwave radiation in the biological material. Theintermittent supply of drying energy may be used to control and regulatethe temperature of biological materials exposed to microwave radiation.In one embodiment, the energy can be delivered according to the dryingkinetics of the specimen itself.

First period of time, as used herein, refers to a preset period of time.Second period of time, as used herein, refers to a preset period oftime. In one embodiment, the first period of time may refer to a periodwherein a vitrification solution may be exposed to microwave radiationand the second period of time may refer to a period wherein avitrification solution is allowed to rest. In another embodiment, thefirst period of time may refer to a period wherein a vitrificationsolution may be allowed to rest and the second period of time may referto a period wherein a vitrification solution may be exposed to microwaveradiation. In yet another embodiment, the first period of time may referto a 1-120 second period wherein a vitrification solution may be exposedto microwave radiation and the second period of time may refer to a1-120 second period wherein a vitrification solution is allowed to rest.

Desiccation energy, as used herein, refers to the energy required to dryout a material and/or solution to a desired level of moisture. In oneembodiment, desiccation energy may refer to energy from a radiant heatsource. In another embodiment, desiccation energy may refer to energysupplied to a dry box. In yet another embodiment, desiccation energy mayrefer to energy supplied by a vacuum chamber.

One embodiment of the present invention discloses a method forpreserving biological material comprising the steps of: a. providing twoor more vitrification solutions, each being comprised of the biologicalmaterial and a vitrification agent, which are in the range from 0.1° C.to 26° C.; b. microwaving the vitrification solutions within the samemicrowave for a first period of time; c. allowing three vitrificationsolutions to rest for a second period of time; d. repeating steps b andc until each vitrification solution enters into a glassy state. Inanother embodiment of the present invention, the vitrification agent iswater-soluble. In still another embodiment, the vitrification agent istrehalose. In yet another embodiment, each vitrification solution iscomprised of a biological material selected from the group comprisingproteins, cells, tissues, organs, cell-based constructs, whole blood,red blood cells, white blood cells, platelets, viruses, bacteria, algae,fungi, sperm cells, spermatocytes, oocytes, ovum, embryos, germinalvesicles, or combinations thereof. In still another embodiment, thefirst period of time and the second period of time are equivalent, notequivalent, or a combination thereof. In yet another embodiment, thefirst period of time and the second period of time are in the range from1 second to 120 seconds. In still another embodiment, the temperature ofthe vitrification solution does not exceed 50° C. for biological cellsand tissues, or above the temperature of thermal denaturation forproteins. In yet another embodiment, the glassy state vitrificationsolution may be stored above cryogenic temperature. In still anotherembodiment, the vitrification solution has a glass transitiontemperature in the range of −150° C. to 30° C. In another embodiment,any of the above methods may further comprise the step of: supplyingadditional desiccation energy from supplemental heat transfer sources.

One embodiment of the present invention discloses a method forpreserving biological material comprising the steps of: a. providing avitrification solution comprised of the biological material and avitrification agent, which is in the range from 0.1° C. to 26° C.; b.microwaving the vitrification solution at a wattage in the range of 1 to600 W for a first period of time until the vitrification solution entersinto a glassy state. In another embodiment of the present invention, thevitrification agent is water-soluble. In still another embodiment, thevitrification agent is trehalose. In yet another embodiment, eachvitrification solution is comprised of a biological material selectedfrom the group comprising proteins, cells, tissues, organs, cell-basedconstructs, whole blood, red blood cells, white blood cells, platelets,viruses, bacteria, algae, fungi, sperm cells, spermatocytes, oocytes,ovum, embryos, germinal vesicles, or combinations thereof. In stillanother embodiment, the first period of time is in the range from 1second to 120 minutes. In still another embodiment, the temperature ofthe vitrification solution does not exceed 50° C. for biological cellsand tissues, or above the temperature of thermal denaturation forproteins. In yet another embodiment, the glassy state vitrificationsolution may be stored above cryogenic temperature. In still anotherembodiment, the vitrification solution has a glass transitiontemperature in the range of −150° C. to 30° C. In yet anotherembodiment, the wattage used is under 200 W. In another embodiment, anyof the above methods may further comprise the step of: supplyingadditional desiccation energy from supplemental heat transfer sources.

21. A method for preserving biological material comprising the steps of:

-   -   a. providing a vitrification solution which is in the range from        0.1° C. to 26° C.; said vitrification solution being comprised        of: said biological material; a vitrification agent;    -   b. microwaving said vitrification solution at a wattage in the        range of 1 to 600 W for a first period of time until said        vitrification solution enters into a glassy state.

22. The method of claim 21 wherein said vitrification agent beingwater-soluble.

23. The method of claim 22 wherein said vitrification agent beingtrehalose.

24. The method of claim 21 wherein said vitrification solution beingcomprised of a biological material being selected from the groupcomprising: proteins, cells, tissues, organs, cell-based constructs,whole blood, red blood cells, white blood cells, platelets, viruses,bacteria, algae, fungi, sperm cells, spermatocytes, oocytes, ovum,embryos, germinal vesicles, or combinations thereof.

25. The method of claim 21 wherein said first period of time being inthe range from 1 second to 120 minutes.

26. The method of claim 21 wherein the temperature of said vitrificationsolution does not exceed 50° C. for biological cells and tissues, orabove the temperature of thermal denaturation for proteins.

27. The method of claim 21 further comprising the step of: supplyingadditional desiccation energy from supplemental heat transfer sources.

28. The method of claim 21 wherein said glassy state vitrificationsolution may be stored above cryogenic temperature.

29. The method of claim 21 wherein said vitrification solution has aglass transition temperature in the range of −150° C. to 30° C.

30. The method of claim 21 wherein the wattage used being under 200 W.

Example Summary

Cell Culture: J774.A1 mouse macrophage cells were obtained from AmericanType Culture Collection (Manassas, Va.). Cultures were maintained at 37°C., 10% CO₂-90% air in cell culture media consisting of Dulbecco'sModified Eagle Medium (DMEM) with 4.5 g/L glucose (Mediatech Inc,Herndon, Va.), 10% fetal bovine serum (FBS; Mediatech Inc, Herndon, Va.)and 1% 5000 I.U. Penicillin, and 5000 μg/ml Streptomycin solution(Mediatech Inc, Herndon, Va.). Cells were first cultured in 25 cm² cellculture T-flasks (Corning Incorporated, NY) for 3 days. As cells nearedconfluence they were detached from flasks by scraping and thentransferred into 50 ml spinner culture flasks (Wheaton Millville, N.J.).All cells used for experiments were taken from established spinner flaskcultures that were maintained at a density of less than 1×10⁶ cells/ml.

Trehalose Loading and Detection: Macrophage cells have the capacity totake up solutes from the extracellular milieu by fluid phaseendocytosis. This approach was used to deliver trehalose into theintracellular space. Cells were suspended in regular culture media thatwas supplemented with 50 mM trehalose and then incubated for 18 hours in50 ml spinner culture flasks (Wheaton Millville, N.J.). Followingincubation for the pre-determined time period, an aliquot of cellsuspension was removed and the cells were collected by centrifugation,washed three times in PBS, and then lysed by freeze-thaw in ultra highpurity 18 mOhm water. The lysed solution was centrifuged and thesupernatant collected and prepared for high performance liquidchromatography analysis. An eighteen inch Dionex Garbo PAC HPLC column(Dionex Sunnyvale, Calif.) and ESA Coulochem II electrochemical detector(ESA Chelmsford, Mass.) were used for sugar determination.

Microwave Drying Protocol: High purity, low endotoxin trehalosedihydrate was obtained from Ferro Pfanstiehl (Waukegan, Ill.). Isotonictrehalose solution was prepared by adding 1 part 1×PBS solution(Mediatech Inc, Herndon, Va.) to 2 parts of 300 mM Trehalose solutionprepared in distilled water. This yielded a 200 mM trehalose solutionwith an average osmolality of 308 mOsm. The osmolality of solutions wasmeasured using a micro-osmometer (Fiske Associates, Norwood, Mass.).

An aliquot of 5 ml of cell suspension was collected from spinner flaskculture and centrifuged at 175×g for 5 min using a Centra CL2 Centrifuge(Thermo Electron Corp., FL). The supernatant was decanted and the cellpellet was resuspended in 1.0 ml of fully complemented Dulbecco'sModified Eagle medium for cell counting in a hemacytometer (HausserScientific, Horsham, Pa.), using 0.4% Trypan Blue (Sigma-Aldrich, MO)exclusion as an indicator of membrane integrity and viability. Based onthis count a cell concentration of 5×10⁵ viable cells/ml in fullcomplement media was prepared. Cells were plated onto 22 mm squareplastic cover slips (Fisher Scientific, Pittsburgh, Pa.) in 20 μldroplets (˜10,000 cells/droplet). The cover slips was placed in 35 mm×10mm tissue culture treated cell culture dishes (Corning Incorporated, NY)and placed inside the incubator for 45 min to allow the cells to attachto the surface of the coverslip. Following the incubation period themedia was carefully removed by pipette and an equal volume of 200 mMtrehalose-PBS drying solution was placed on top of the plated cells.This sample was then placed in a single phase 120 volt 2450 MHzMicrowave oven (Danby, Ontario, Canada; input specification: 60 Hz AC,1.05 KW, grounded). This unit has a maximum output power of 600 W.

To avoid over-heating the samples, several heating processes wereevaluated. Samples were heated in steps, alternating between activeheating steps at full power, and passive cooling steps, wherein thesample was allowed to rest inside the microwave cavity with the dooropen. This process facilitated acquisition of temperature data betweenheating periods. Active heating periods between 15 and 45 seconds wereevaluated. The passive cooling step was held constant at 30 s.Immediately after the power was terminated in each heating step, themicrowave door was opened, a K type thermocouple was embedded into thecentral region of the droplet, (Omega Engineering Inc., Stamford, Conn.)and the temperature was recorded on a single input printing thermometer(Model 08533-41, Cole Parmer, Vernon Hills, Ill.), Samples were heateduntil the sample appeared glassy or solid-like to the eye.

Using the optimized heating profile, mass loss curves as a function ofcumulative microwave heating time were generated. The initial and finalweights were determined and used to calculate moisture contents (gramsof water per gram of dry weight). The dry weight of the sample was foundby preparing parallel samples in the same manner and drying them at 110°C. for 24 hours in a vacuum oven, in replicates of 6.

The viability of microwave-processed cells was evaluated as a functionof final moisture content. Immediately after microwave processing thecoverslip was removed from the microwave and placed into a P-35 cellculture dish. It was then rehydrated with 100 μl of fully complementedDulbecco's Modified Eagle medium that had been pre-warmed to 37° C.Samples were then placed in the incubator for 45 minutes to provide timefor reattachment to the surface of the coverslip prior to viabilityassessment.

Viability Assessment: Cell viability was determined using Trypan Blueand Calcein-AM/Propidium Iodide membrane integrity assays (MolecularProbes, Eugene, Oreg.). The stock solution for the Calcein-AM/PropidiumIodide staining was prepared by adding 10 μl of 1 mg/ml Calcein AMsolution (aq.) and 5 μl of 1.0 mg/ml solution Propidium Iodide solution(aq.) to 10 ml of Phosphate-buffered Saline (Mediatech Inc, Herndon,Va.). Following the recovery period in the incubator, the entire 100 μldroplet was gently removed (leaving behind the cells which were attachedto the coverslip surface) and collected into a 0.5 ml eppendorf tube.The viability of detached cells in this aliquot was determined bystaining with 0.4% Trypan Blue solution and counting in a hemacytometer(Hausser Scientific, Horsham, Pa.). An aliquot of 120 μl ofCalcein-AM/Propidium Iodide solution in PBS was added to the attachedcells on the coverslip and the sample was then incubated at 37° C. for 5min. These samples were then imaged using an inverted researchmicroscope (Olympus Biosystems 1×81; Olympus America Inc., Melville,N.Y.) with FITC and PI fluorocubes. The attached cell viability wasdetermined by counting the live (green) and dead (red) cells on threerepresentative images from the same sample. The total viability wasestimated by the following formula which takes into consideration theviability of both detached and attached cells:

${{Total}\mspace{14mu}{Viability}} = {\frac{{Number}\mspace{14mu}{of}\mspace{14mu}{viable}\mspace{14mu}{detached}\mspace{14mu}{cells}}{10,000} + {\frac{{10,000} - {{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{detached}\mspace{14mu}{cells}}}{10000} \cdot \left( {{Fractional}\mspace{14mu}{Viability}\mspace{14mu}{of}\mspace{14mu}{attached}\mspace{14mu}{cells}} \right)}}$

Long term viability of the microwave processed cells was determinedusing a metabolic assay based on the reduction of resazurin. The blueand non-fluorescent dye resazurin is reduced to pink and highlyfluorescent resorufin by the metabolic activity of living cells.Resazurin was obtained in powdered form (Sigma Aldrich Co., Milwaukee,Wash.). A stock solution of 110 μg/ml was prepared in phosphate-bufferedsaline (Mediatech Inc, Herndon, Va.), sterile-filtered, and then storedin the dark at 4° C. Microwave processed cell specimens on coverslipswere placed inside the wells of a 24-well plate (Corning Incorporated,NY), rehydrated with fully complemented media, and then placed insidethe incubator at 37° C. Metabolic function assessment was performedafter 4-, 8-, 18.75-, 24-, 30-, 42-, 48-, and 54.5-hour recoveryperiods. One hour prior to the assay point cell cultures were removedfrom the incubator, the media removed, and 500 μL of phenol red-freefully complemented medium with 10% resazurin solution was added to eachwell. Following the addition, the plates were incubated for 60 minutesat 37° C. in the dark. After incubation, the cell viability was assessedin terms of Arbitrary Fluorescence Units (AFU) using a fluorescent platereader (Synergy HT, Bio-Tek Instruments, VT) with a 530 nm excitationand a 590 nm emission filter set. Following each measurement, theresazurin solution was removed from the wells, replaced with 500 ml offull-complement culture medium, and the plates were returned to theincubator.

The distribution of the cells within the dried droplets were determinedusing both brighffield and fluorescence microscopy. Calcein-AM andPropidium Iodide stains were used for fluorescence live-dead staining ofthe cells as described previously and images were acquired in variousregions that spanned the droplet. For comparison purposes equivalentsamples were also dried to the same moisture content using aconventional desiccation chamber and imaged in the same manner.Macroscopic differences in the distribution of solids were noted by eye,and representative brightfield images were obtained to demonstrate thedifferences in drying characteristics by the two different techniques.

Results

Process Optimization: Cells that were incubated in trehalose solutionfor 18 hours were determined to have loaded 160 mM±50 mM trehaloseduring this time, a portion of which would be expected to be distributedin the cytoplasm, and the remainder residing in the endosomes.

In FIG. 1 the temperature at the interior of the droplet was plotted asa function of cumulative microwave heating time for several differentactive heating periods. As expected, the extent of heating was found tobe directly proportional to the length of microwave processing at fullpower (600 Watts). The level of dryness that was achieved by the end ofthe process is shown in the brackets, expressed in units of gH₂O/gdw.Because of the dipole heating action of the microwave energy thetemperature of the samples can increase quickly, thus moderate activeheating periods are necessary to avoid large thermal excursions. Becauseof the nature of the temperature measurement procedure, some delay(seconds) in acquiring the temperature is unavoidable. As such, therecorded temperature represents a low estimate of the true temperature.Furthermore the temperature also represents an average value for theinterior portion of the droplet, and the spatial positioning can beconsidered accurate to only a few millimeters. The utility in thismeasurement is in the delineation of a reasonable heating profile thatavoids a persistent excursion above 50° C. Above this temperature theinduction of a heat shock response is likely in the cells. An activeheating period of 15 or 30 seconds did not result in a recordedtemperature above typical physiological temperatures, whereassignificant heating was observed for 45 second active heating periods.

Drying Characteristics: Microwave treatment of the cell samples resultedin a systematic water loss from the sample as shown in FIG. 2. Theweight loss data is represented in grams of water per gram of dry weightof the samples and the weight measurements were obtained using a highprecision analytical balance (Mettler Toledo AX Series AnalyticalBalance, Columbus, Ohio). The drying rate was observed to be influencedby the local relative humidity; hence values of relative humidity areincluded with each data set.

The characteristic cell survival 45 minutes after microwave exposure wasdetermined using standard viability assays. Membrane integrity wasdetermined using Trypan Blue staining on detached cells and Calcein-AMand Propidium Iodide staining for attached cells. The graph in FIG. 3shows the total viability (calculated using equation 1) of the microwavetreated cells that do not have exogenous intracellular protection. Theviability of the detached fraction of cells is also shown. Both of thesedata sets follow a similar trend. The inset graph also shows the totalnumber of detached cells at different levels of dryness during themicrowave drying process. As the sample gets progressively drier, thenumber of cells that remain attached to the surface upon rehydrationdecreases. At the lowest moisture levels almost all of the plated cellsare detached upon rehydration. The graph in FIG. 4 displays similarviability data for cells that had been previously incubated withintracellular trehalose prior to the microwave drying process. Asexpected, cells that accumulated intracellular trehalose demonstrated agreater tolerance of moisture loss than the control cells. This trend isconsistent with other drying modalities and demonstrates the osmoticprotection provided by trehalose. The inset graph also demonstrates asimilar relationship with moisture content as the control cells, withthe number of detached cells upon rehydration increasing as the dryingprogresses, with the exception that more variability in the data isobserved at low moisture contents.

In FIG. 5 the fluorescence emission of resazurin is shown as a functionof cell culture time. The metabolism of resazurin is indicative of thetotal number of metabolically active cells and hence increasingfluorescence over time generally demonstrates growth of cells. Cellsamples that had been previously incubated in trehalose for 18 hourswere microwaved for an accumulative time of 420 s (30 s active/30 srest) as described previously. An initial concentration of 10,000 cellsper droplet was used. After 48 hours the fluorescence signal inmicrowave processed cells was 64.8% of the signal in control cells. Thisfraction was consistent with the number of living cells in the sample 45minutes after the treatment, as assessed by Calcein-AM/propidium iodidestaining.

FIG. 6 shows micrographs of trehalose-loaded cells dried in droplets byboth the microwave technique and by processing in a drybox. In both ofthese cases the samples were dried to the same moisture content based ona bulk gravimetric measurement (4.258 gH₂O/gdw). Non-uniformity incellular distribution was obvious by eye in samples dried in thedesiccation chamber. The central region of the droplet, which wasclearly wetter than the periphery, contained a white cluster of solids.Microscopy confirmed this as clumped cellular material. Few cells wereobserved at the periphery. Furthermore a ring of solids could beobserved in the periphery, consistent with drying physics thatcharacterize a contact-pinned droplet. This effect was not observed insamples that were dried by microwave. In the microwave processed samplescells are found to be uniformly distributed throughout the sample.Because of the dramatic differences in the distribution of cells in thesamples dried by drybox and microwave technique no rigorousquantification technique was used to distinguish the two samples.

FIG. 7 contains micrographs of samples dried by both techniques,rehydrated, and then stained with Calcein AM/PI solution. The sampleswere dried to the same moisture content based on a bulk gravimetricmeasurement (4.258 gH₂O/gdw). Consistent with brightfield images,micrograph 7.A clearly show the cluster of cells at the center of adroplet that was dried in a drybox, and the relatively few cells at theedges of the droplet (7B). The clustering of cells precluded aquantitative analysis of viability. As shown in the previousmicrographs, the clustering effect is not observed in the case ofmicrowave drying wherein cells are found to be more uniformlydistributed throughout the sample. No trend of viability with locationwas observed in either case.

EXAMPLES Example 1 Control Cells Dried in Isotonic Trehalose Solution

-   Cell culture. Mouse macrophage cells were obtained from American    Type Culture Collection (Manassas, Va.). Cultures were maintained at    37° C., 10% CO₂-90% air in cell culture media consisting of    Dulbecco's modified Eagle medium (DMEM) with 4.5 g/L glucose    (Mediatech Inc, Herndon, Va.), 10% fetal bovine serum (FBS;    Mediatech Inc, Herndon, Va.) and 1% 5000 I.U. Penicillin & 5000    ug/ml Streptomycin solution (Mediatech Inc, Herndon, Va.). Cells    were first cultured in 25 cm² cell culture T-flasks (Corning    Incorporated, NY) for 3 days. As cells neared confluence they were    detached from the flasks by scraping and then transferred into 50 ml    spinner culture flasks (Wheaton Millville, N.J.). An aliquot of    cells was taken from an established spinner flask cultures that was    maintained at a density of less than 1×10⁶ cells/ml.-   Cell Processing. An aliquot of 5 ml of cell suspension was collected    from spinner flask culture and centrifuged at 175×g for 5 min using    a Centra CL2 Centrifuge (Thermo Electron Corp., FL). The supernatant    was decanted and the cell pellet was resuspended in 1.0 ml of fully    complemented Dulbecco's Modified Eagle medium for cell counting in a    hemacytometer (Hausser Scientific, Horsham, Pa.), using 0.4% Trypan    Blue (Sigma-Aldrich, MO) exclusion as an indicator of membrane    integrity and viability. Based on this count a cell concentration of    5×10⁵ viable cells/ml in full complement media was prepared. Cells    were plated onto 22 mm square plastic cover slips (Fisher    Scientific, Pittsburgh, Pa.) in 20 μl droplets (˜10,000    cells/droplet). The cover slips was placed in 35 mm×10 mm tissue    culture treated cell culture dishes (Corning Incorporated, NY) and    placed inside the incubator for 45 min to allow the cells to attach    to the surface of the coverslip.-   Cell Drying. Following the incubation period the media was carefully    removed by pipette and an equal volume of 200 mM isotonic    trehalose-PBS drying solution was placed on top of the plated cells    (total osmolality=308 mOsm). This sample was then placed in a single    phase 120 volt 2450 MHz Microwave oven for processing (Danby,    Ontario, Canada; input specification: 60 Hz AC, 1.05 KW, grounded).    Samples were heated in steps, alternating between 30 s active    heating steps at full power, and passive cooling steps, wherein the    sample was allowed to rest inside the microwave cavity with the door    open (30 s). Samples were dried to a final moisture content of 4.258    gH2O/gdw.-   Cell Survival: The viability of microwave-processed cells was    evaluated as a function of final moisture content. Immediately after    microwave processing the coverslip was removed from the microwave    and placed into a P-35 cell culture dish. It was then rehydrated    with 100 μl of fully complemented Dulbecco's Modified Eagle medium    that had been pre-warmed to 37° C. Samples were then placed in the    incubator for 45 minutes to provide time for reattachment to the    surface of the coverslip prior to viability assessment. Cell    viability was determined using Trypan Blue and Calcein-AM/Propidium    Iodide membrane integrity assays (Molecular Probes, Eugene, Oreg.).    Following the recovery period in the incubator, the entire 100 μl    droplet was gently removed (leaving behind the cells which were    attached to the coverslip surface) and collected into a 0.5 ml    eppendorf tube. The viability of detached cells in this aliquot was    determined by staining with 0.4% Trypan Blue solution and counting    in a hemacytometer (Hausser Scientific, Horsham, Pa.). An aliquot of    120 μl of Calcein-AM/Propidium Iodide solution in PBS was added to    the attached cells on the coverslip and the sample was then    incubated at 37° C. for 5 min. These samples were then imaged using    an inverted research microscope (Olympus Biosystems 1×81; Olympus    America Inc., Melville, N.Y.) with FITC and PI fluorocubes. The    attached cell viability was determined by counting the live (green)    and dead (red) cells on three representative images from the same    sample. The total viability was estimated using equation 1    (described previously), which takes into consideration the viability    of both detached and attached cells. The percentage of cells that    were viable after processing to this moisture content was    approximately 18%.

Example 2 Trehalose Pre-treated Cells Dried in Isotonic TrehaloseSolution

-   Cell culture. Same as Example 1-   Cell Processing. Mouse macrophage cells were incubated overnight in    full complement media containing trehalose at a concentration of 50    mM. Cells were found to load trehalose at a concentration of 160±50    mM (standard error), some of which was expected to be distributed in    the cytoplasm. Cells were then dried to a final moisture content of    4.258 gH2O/gdw using the processing method of Example 1.-   Cell Drying. Same as Example 1-   Cell Survival: The method for assessing cell viability was the same    as in Example 1. The percentage of cells that were viable after    processing to 4.258 gH2O/gdw was approximately 70%.-   Cell growth: Long term viability of the microwave processed cells    was determined using a metabolic assay based on the reduction of    resazurin. The blue and non-fluorescent dye resazurin is reduced to    pink and highly fluorescent resorufin by the metabolic activity of    living cells. Resazurin was obtained in powdered form (Sigma Aldrich    Co., Milwaukee, Wash.). A stock solution of 110 μg/ml was prepared    in phosphate-buffered saline (Mediatech Inc, Herndon, Va.),    sterile-filtered, and then stored in the dark at 4° C. Microwave    processed cell specimens on coverslips were placed inside the wells    of a 24-well plate (Corning Incorporated, NY), rehydrated with fully    complemented media, and then placed inside the incubator at 37° C.    Metabolic function assessment was performed after 4, 8, 18.75, 24,    30, 42, 48, and 54.5 hour recovery periods. One hour prior to the    assay point cell cultures were removed from the incubator, the media    removed, and 500 μL of phenol red-free fully complemented medium    with 10% resazurin solution was added to each well. Following the    addition, the plates were incubated for 60 min at 37° C. in the    dark. After incubation, the cell viability was assessed in terms of    Arbitrary Fluorescence Units (AFU) using a fluorescent plate reader    (Synergy HT, Bio-Tek Instruments, VT) with a 530 nm excitation and a    590 nm emission filter set. Following each measurement, the    resazurin solution was removed from the wells, replaced with 500 ml    of full-complement culture medium, and the plates were returned to    the incubator. At 48 hours the fraction of viable metabolically    active cells in the microwave-treated sample was 64.8%.    Sample Uniformity:

The distribution of the cells within the dried droplets were determinedusing both brightfield and fluorescence microscopy. Calcein-AM andPropidium Iodide stains were used for fluorescence live-dead staining ofthe cells as described previously and images were acquired in variousregions that spanned the droplet. Representative brightfield images wereobtained. By eye, there appeared to be an even distribution of solidsthroughout the sample. The characteristic ring observed in samples driedby passive techniques was not seen in the microwaved samples. Microscopyrevealed an even distribution of cells throughout the sample.

Example 3 Control Cells Dried in Hypertonic Trehalose Solution

-   Cell culture. Same as Example 1-   Cell Processing. Same as Example 1-   Cell Drying. Same as Example 1 with the following exception:

Following the incubation period the media was carefully removed bypipette and an equal volume of 200 mM hypertonic trehalose-PBS dryingsolution was placed on top of the plated cells (total osmolality=508mOsm).

-   Cell Survival: The method for assessing cell viability was the same    as in example 1. The percentage of cells that were viable after    processing to 4.258 gH2O/gdw was approximately 34%.

Example 4 Trehalose Pre-Treated Cells Dried in Hypertonic TrehaloseSolution

-   Cell culture: Same as Example 1-   Cell Processing: Same as Example 2-   Cell Drying. Same as Example 3-   Cell Survival: The method for assessing cell viability was the same    as in example 1. The percentage of cells that were viable after    processing to 4.258 gH2O/gdw was approximately 54%.

The present invention may be embodied in other forms without departingfrom the spirit and the essential attributes thereof, and, accordingly,reference should be made to the appended claims, rather than to theforgoing specification, as indicated in the scope of the invention.

Example 5

200 milliliters of a 300 mM Trehalose (Ferro Pfansteihl, Cleveland Ohio)solution in 18.2 MO deionized water was mixed with 100 milliliters of1×PBS (Mediatech Inc., Manassas, Va.) to produce a solution with ameasured osmolality of 316 mmol/kg (±3.08). Osmolality measurements weremade using a Vapro 5520 (Wescor Inc., Logan, Utah) vapor pressureosmometer. The instrument was standardized using 100, 290, and 1000mmol/kg sodium chloride standards (Wescor Inc., Logan, Utah) with thesample measured five times to produce an average and standard deviation.For both experiments a 600 W microwave (Danby Inc., Ontario, Canada) wasused. The glass turntable was removed and the sample was placed directlyon the center of the drive mechanism that rotates the turntable duringmicrowaving.

For the temperature versus time data (FIG. 8), a 20 μL aliquot oftrehalose solution was pipette on a cover slip which was placed in aculture dish. The covered culture dish was refrigerated for severalhours and was then placed in the microwave, the cover was removed and athermocouple attached to Fluke Hydra Series II data acquisition unit(John Fluke Corporation, Everett, Wash.). The microwaving periods weremeasured at 15 and 30 second heating cycles. The temperature wasmeasured after each heating cycle after a passive 30 second coolingperiod.

For the mass loss experiments (FIG. 9) a 22 mm plastic cover slip(Fisher Scientific) was placed in a 35 mm×10 mm (P-35) cell culturedishes (Corning Inc., Corning N.Y.) and weighed on an AX105 Delta Range®analytical balance (Mettler Toledo Inc., Columbus, Ohio). A 20 μLaliquot of trehalose solution was then pipetted on the cover slip in theculture dish and re-weighed. This mass was recorded as the zero pointfor the microwave experiment. The samples were then microwaved for a 30second interval with a 30 second cooling periods between each heatingcycle. The mass was measured after increments of cumulative microwavingof approximately 100 seconds. The samples were then placed in a vacuumoven to ensure complete drying. The mass of the dry samples were used indetermining the amount of water lost during the microwave process. Therelative humidity was measured using a pen type thermo-hydrometer(Control Co., Friendswood, Tex.)

Example 6 Preservation of Multiple Samples Simultaneously

The objective of this experiment was to develop a microwave dryingprocess and associated apparatus to enable the removal of water frommultiple small volume biological samples, while maintaining theirtemperature below 50° C. This experiment required the following:

-   -   1) The simultaneous processing of multiple samples.    -   2) That samples are dried and stored in the same container.    -   3) That reusable drying devices be used.    -   4) Sterile processing    -   5) The use of microwave-safe components.

Cells were pre-loaded with a trehalose solution (anhydrous protectant)which were then deposited in 10 μL droplets on a polycarbonate membrane.The membrane is then placed within a container and the container is thenplaced within a SAM 255 microwave. The cells were then dried using a SAM255 microwave at low wattage (120 W) for a continuous amount of time.The polypropylene containers are enclosed after drying is complete andstored in individual vacuum packs in dry storage.

Looking to FIG. 10 we see illustrated the decrease in the grams of H₂Oper grams dry weight over time (minutes) for the trehalose solution.Looking now to FIG. 11 we see that the method used in this examplemaintains a temperature well below 50° C. and thus avoids damaging thebiological material within the vitrification solution.

Example 7 Preservation of Reproductive Cells and Tissues

The objective of this experiment was to assess the impact of membraneporation (using hemolysin), trehalose exposure and desiccation on GVchromatin structure. Grade 1 cat oocytes (n=192) were denuded andexposed to 1.0 mM resveratrol. Oocytes were incubated in 0 or 10 μg/mlhemolysin for 20 min (38.5° C.) and then exposed to a solution of 0 or40% (w/w) trehalose for 15 minutes at room temperature. After each ofthe four conditions, half the oocytes were assessed for trehalosepenetration (fluorescence anisotropy measurements), GV chromatinstructure (Hoechst staining) and DNA integrity (fragmentation detectedusing a Comet assay). Remaining oocytes were deposited on a 10 μl drop(0 or 40% trehalose) and desiccated using 30 sec microwave pulses (600W, 2450 MHz) every 30 sec (up to a total of 15 pulses). After eachpulse, samples were weighed on a micro-weight balance to monitor changesin water content. When 0.2 g of water/g of dry weight was achieved,samples were rehydrated to examine GV chromatin structure and extent ofDNA integrity (see FIG. 12). Minimum water content and a highly viscoustrehalose state were reached in 450 seconds of cumulated microwavepulses. GV chromatin structure and DNA stability were unaffected afterporation and trehalose exposure alone (Table 1).

TABLE 1 Qualitative assessment of germinal vesicle structure and DNAintegrity after treatments with or without membrane poration, trehaloseexposure and desiccation. Treatments Membrane Trehalose Chromatin DNAporation exposure Desiccation n structure n integrity − − − 11 Normal 12Yes − + − 10 Normal 11 Yes + − − 9 Normal 11 Yes + + − 10 Normal 10 Yes− − + 11 Degenerate 11 No − + + 12 Normal 10 No + − + 11 Degenerate 12No + + + 10 Normal 11 Yes

Oocytes treated with hemolysin and then exposed to 40% trehalose did notshrink as a result of exposure to hyper-osmotic solution (FIG. 13B)compared to what was observed in the absence of membrane poration (FIG.13C). After membrane poration and trehalose exposure, anisotropymeasurements confirmed the absence of a difference between theextracellular and intracellular fluorescence, which means that trehalosehomogeneously penetrated the porated oocytes as well as the GV. Resultsassociated with GV chromatin structure and DNA stability afterdesiccation are presented in Table 1 and illustrated in FIG. 13. Withporation, but in the absence of trehalose before desiccation, the GVchromatin structure degenerated (FIG. 13D) and the DNA fragmented (FIG.13G). However, with poration and exposure to 40% trehalose, both GVstructure (FIG. 13) and integrity (FIG. 13) were retained.Interestingly, without membrane poration, but in the presence oftrehalose before desiccation, GV structure was maintained (FIG. 13F),but DNA was fragmented (FIG. 13I). These results clearly demonstratedour ability to incorporate a natural lyoprotectant, trehalose, into theoocyte cytoplasm and GV. Furthermore, and most importantly, this processallowed GV structure and DNA integrity to be preserved, even aftermicrowave processing for desiccation.

1. A method for preserving a biological material comprising the stepsof: a. providing a vitrification solution which is in the range from0.1° C. to 17.9° C.; said vitrification solution being comprised of saidbiological material and a vitrification agent; b. microwaving saidvitrification solution for a first period of time; c. allowing saidvitrification solution to rest for a second period of time; d. repeatingsteps b and c until said vitrification solution enters into a glassystate.
 2. The method of claim 1 wherein said vitrification agent iswater-soluble.
 3. The method of claim 2 wherein said vitrification agentis trehalose.
 4. The method of claim 1 wherein said biological materialis selected from the group comprising proteins, cells, tissues, organs,cell-based constructs, whole blood, red blood cells, white blood cells,platelets, viruses, bacteria, algae, fungi, sperm cells, spermatocytes,oocytes, ovum, embryos, germinal vesicles, or combinations thereof. 5.The method of claim 1 wherein said first period of time and said secondperiod of time are equivalent, not equivalent, or a combination thereof.6. The method of claim 1 wherein said first period of time and saidsecond period of time are in the range from 1 second to 120 seconds. 7.The method of claim 1 wherein the temperature of said vitrificationsolution does not exceed 50° C. for biological cells and tissues, orabove the temperature of thermal denaturation for proteins.
 8. Themethod of claim 1 further comprising the step of supplying additionaldesiccation energy from supplemental heat transfer sources.
 9. Themethod of claim 1 wherein said glassy state vitrification solution maybe stored above cryogenic temperature.
 10. The method of claim 1 whereinsaid vitrification solution has a glass transition temperature in therange of −150° C. to 30° C.
 11. A method for preserving biologicalmaterials comprising the steps of: a. providing two or morevitrification solutions which are in the range from 0.1° C. to 26° C.;said vitrification solutions each being comprised of said biologicalmaterial and a vitrification agent; b. microwaving said vitrificationsolutions within a microwave oven for a first period of time; c.allowing said vitrification solutions to rest for a second period oftime; d. repeating steps b and c until each said vitrification solutionsenter into a glassy state.
 12. The method of claim 11 wherein saidvitrification agent is water-soluble.
 13. The method of claim 12 whereinsaid vitrification agent is trehalose.
 14. The method of claim 11wherein each vitrification solution is comprised of the biologicalmaterial selected from the group comprising proteins, cells, tissues,organs, cell-based constructs, whole blood, red blood cells, white bloodcells, platelets, viruses, bacteria, algae, fungi, sperm cells,spermatocytes, oocytes, ovum, embryos, germinal vesicles, orcombinations thereof.
 15. The method of claim 11 wherein said firstperiod of time and said second period of time are equivalent, notequivalent, or a combination thereof.
 16. The method of claim 11 whereinsaid first period of time and said second period of time are in therange from 1 second to 120 seconds.
 17. The method of claim 11 whereinthe temperature of said two or more vitrification solutions do notexceed 50° C. for biological cells and tissues, or above the temperatureof thermal denaturation for proteins.
 18. The method of claim 11 furthercomprising the step of supplying additional desiccation energy fromsupplemental heat transfer sources.
 19. The method of claim 11 whereinsaid glassy state vitrification solutions may be stored above cryogenictemperature.
 20. The method of claim 11 wherein said two or morevitrification solutions have a glass transition temperature in the rangeof −150° C. to 30° C.